Biosafety classification is based on U.S. Public Health Service Guidelines, it is the responsibility of the customer to ensure that their facilities comply with biosafety regulations for their own country.
Isolation
Hot water tank, California, 1987
Product Format
frozen
Storage Conditions
Frozen Cultures: -70°C for 1 week; liquid N2 vapor for long term storage
Freeze-dried Cultures: 2-8°C
Live Cultures: See Protocols section for handling information
Type Strain
no
Comments
Intracellular multiplication of Legionella pneumophila
Medium
ATCC® Medium 711: PYB
Growth Conditions
Temperature: 25°C
Culture System: Xenic, grown with mixed bacteria
Cryopreservation
Harvest and Preservation
Harvest cells from a culture which is at or near peak density by adding 5 mL fresh ATCC medium 1323 (Page's Balanced Salt Solution) and washing cells into suspension. Rub the surface of the plate with a spread bar to detach adhering amoebae.
Transfer the liquid medium to a sterile centrifuge tube.
If the cell concentration does not exceed 2 x 106 cells/mL adjust the suspension to that concentration. To adjust the concentration, centrifuge at 600 x g for 5 min and resuspend the pellet in the volume of fresh medium required to yield 2 x 106.
While cells are centrifuging prepare a 15% (v/v) solution of sterile DMSO as follows: Add the required volume of DMSO to a glass screw-capped test tube and place it in an ice bath. Allow the DMSO to solidify. Add the required volume of refrigerated medium. Dissolve the DMSO by inverting the tube several times.
*NOTE: If the DMSO solution is not prepared on ice, an exothermic reaction will occur that may precipitate certain components of the medium.
Mix the cell preparation and the DMSO in equal portions. Thus, the final concentration will be at least 106 cells/mL and 7.5% (v/v) DMSO. The equilibration time (the time between addition of DMSO and the start of the cooling cycle) should be no less than 15 min and no longer than 60 min.
Dispense in 0.5 mL aliquots into 1.0 - 2.0 mL sterile plastic screw-capped cryules (special plastic vials for cryopreservation).
Place vials in a controlled rate freezing unit. From room temperature cool at -1°C/min to -40°C. If freezing unit can compensate for the heat of fusion, maintain rate at -1°C/min through heat of fusion. At -40°C plunge ampules into liquid nitrogen.
The frozen preparations are stored in either the vapor or liquid phase of a nitrogen freezer.
To establish a culture from the frozen state place an ampule in a water bath set at 35°C (2-3 min). Immerse the vial to a level just above the surface of the frozen material. Do not agitate the vial.
Immediately after thawing, aseptically remove the contents of the ampule and distribute to the center of a fresh plate of ATCC medium 711. Distribute the material evenly over the plate using a spread bar. Incubate at 25°C.
Name of Depositor
BS Fields
Year of Origin
1987
References
Fields BS, et al. Intracellular multiplication of Legionella pneumophila in amoebae isolated from hospital hot water tanks. Curr. Microbiol. 18: 131-137, 1989.
